PLANT DISEASE DIAGNOSIS - The Epidemiology of Plant Diseases - page 27

uncharacterized genotypes, but for commercial purposes DNA chips are used
in SNP identification. A DNA chip is suitable for scoring several SNPs in parallel
from each sample in a multiplexed fashion. DNA chips have immobilized
oligonucleotides of known sequences that differ at specific sites of individual
nucleotides and are used for SNP detection. This makes use of the technique
'sequencing by hybridization' and involves tiling strategy. Four oligonucloetides in
a column of an array will differ only at the SNP site and only one will be fully
homologous. When such an array is hybridized with biotinylated PCR product, the
perfect match will allow binding and mismatched products will be washed away.
The perfect match in each case can be detected by using this detection system
(Gupta et al., 2001). SNPs can also be detected by minisequencing using Sanger's
dideoxynucleotide method, where the oligonucleotide primer has a sequence one or
more bases upstream of the SNP site. A mixture of all the four dNTPs and one of the
four possible ddNTPs that corresponds to the SNP locus, is used for primer
extension, so that the incorporation of a single ddNTP at the SNP site will terminate
the reaction and will allow detection of the SNP. A new sequencing method called
Pyrosequencing has been developed to obtain short DNA sequences for SNP
detection. This method relies on the step-wise addition of individual dNTP (with
simultaneous release of pyrophosphates) and monitoring their template guided
incorporation into the growing DNA chain via chemiluminescent detection of the
formation of pyrophosphates (Ronaghi and Elahi, 2002). The unincorporated dNTP
will be degraded using the enzyme apyrase. The pyrophosphate released is utilized
to convert 5' amino phosphosulfurate into ATP with the help ATP sulfurylase, and
the ATP produced drives luciferase-mediated conversion of luciferin into
oxyluciferin, generating light. The light produced is proportionate to the ATP
released, which in turn is directly proportional to dNTP consumption. The emitted
light is detected by a charge coupled device camera and seen in a programme as
a peak; whole height will reveal the number of dNTP molecules consumed.
Pyrosequencing is particularly suitable for SNP genotyping as genotyping of
previously identified SNPs by this method requires sequencing of only a few
nucleotides (Gupta et al., 2001).
Kroon et al. (2004) distinguished isolates of Phytophthora ramorum from
Europe and America based on a point mutation in the mitochondrial Cox gene by
developing a single nucleotide polymorphism-based method in which a multiple
copy marker was amplified in P. ramorum isolates; this was later digested with the
restriction enzyme Apo I. SNP in the cytochrome b gene has been found to confer
resistance to strobilurin fungicides in Blumeria ( Erysiphe) graminis f.sp. tritici .
Based on this point mutation, three different types of molecular markers have been
developed by Baumler et al. (2003) to score resistant and sensitive isolates from
specifically selected regional populations across Europe. The results of molecular
tests were in complete concordance to those revealed by in vivo tests, indicating the
potential of SNP applications on a large scale.
DNA microarrays offer the latest technological advancement for multi-gene
detection and diagnostics (Call, 2001). DNA microarrays were first described by
Schena et al. (1995) for the simultaneous analyses of large-scale gene expressions
by a large number of genes (Schena et al., 1996; Lochhart et al., 1996; DeRisi et al.,